Systems and methods for autologous biological therapeutics

ABSTRACT

An autologous cell concentrating system and method are disclosed. The system has a blood separation component, a first vessel, a second vessel, a first valve, a second valve, and a concentration and flow logic and control component. The concentration and flow logic and control component is configured to: determine a first volume of a target cell-poor fraction in the first vessel to mix with a target cell-rich fraction in the second vessel in order to form a target cell-rich concentrate having a concentration of target cells that is within a target concentration range; and control the second valve to transfer the first volume of the target cell-rich fraction from the first vessel to the second vessel to form the target cell-rich concentrate. The target concentration range is between 1.0 and 1.5×106 target cells/μL.

FIELD OF THE INVENTION

This application is a continuation of U.S. application Ser. No.13/421,728 filed Mar. 15, 2012 and entitled “SYSTEMS FOR AUTOLOGOUSBIOLOGICAL THERAPEUTICS,” which claims priority to U.S. PatentApplication Ser. No. 61/453,658, filed Mar. 17, 2011 and entitled“SYSTEMS AND METHODS FOR PRODUCING A PLATELET RICH PLASMA,” all of whichis incorporated herein by reference in its entirety and for allpurposes.

FIELD OF THE INVENTION

The present invention relates to systems, methods, and apparatuses forautologous biological therapeutics, including but not limited toplatelet rich plasma (PRP) and bone marrow cell concentrate (BMCC)therapy and technology.

BACKGROUND OF THE INVENTION

One of the proven methods of enhancing hard and soft tissue regenerationis the addition of human growth factors to a wound site or surgicalincision. A safe and simple way of procuring compatible growth factorsin a clinical situation is by the isolation of platelets from the bloodof the patient, referred to as an “autologous platelet concentrate.”

Platelets are blood cells primarily involved in arresting bleeding.However, they also contain proteins called growth factors that helppromote healing and tissue regeneration. Man-made highly concentratedmixtures of platelets (platelet concentrates or Platelet rich plasma(PRP)) have higher platelet counts than natural blood and have beenfound to stimulate the body's soft tissue and hard tissue regeneration.

Bone marrow cell concentrate (BMCC) may include a number of target cellsincluding the following: stem-like cells (pluripotent cells) (e.g.,monocytes), white blood cells, platelets, neutrophils, lymphocytes,eosinophils, and basophils, all of which have a variety of uses inhealing, regeneration, and treatment. Since it is difficult to separateany one or more of these cell fractions from a BMCC, they are typicallyall inserted or injected into a patient. In one embodiment, the BMCCincludes platelets and white blood cells, including a stem cellfraction, where the stem cell fraction enhances the regenerative effectsof the platelets.

Improved understanding of the role of growth factors as biochemicalmediators of wound healing has paved the way for a new family ofbioactive therapeutic products to expedite wound healing. Delivery ofgrowth factors (recombinant or as autologous platelets) has emerged as apossible commercial opportunity for improving the clinical outcomes ofsoft, bone, and connective tissue repair. However, there is inability inthe art to control or manipulate final product concentrations to anarrow target range, required to study or define dose-responserelationships, and ultimately validate therapeutic effectiveness ofthese agents.

While hemoanalysis machines are capable of measuring typical plateletconcentrations, they are not suitable for measuring the high plateletconcentrations found in PRP transfusions. These machines are also largeand expensive (e.g., $15,000-20,000/unit).

Another challenge of forming platelet and BMC concentrations is thatseparation and concentration procedures can prematurely activateplatelets thus starting the clotting cascade. Thus, there is a need forseparation procedures that avoid premature activation of platelets.

SUMMARY

Exemplary embodiments of the present invention that are shown in thedrawings are summarized below. These and other embodiments are morefully described in the Detailed Description section. It is to beunderstood, however, that there is no intention to limit the inventionto the forms described in this Summary of the Invention or in theDetailed Description. One skilled in the art can recognize that thereare numerous modifications, equivalents and alternative constructionsthat fall within the spirit and scope of the invention as expressed inthe claims.

In one aspect, this disclosure describes a cell concentrating system.The system has a blood separation component, a first vessel, a secondvessel, a first valve, a second valve, and a concentration and flowlogic and control component. The blood separation component has a bloodsample input and a centrifuge to separate the blood sample into a redblood cell fraction and a plasma fraction. The plasma fraction has atarget cell-rich fraction and a target cell-poor fraction. The bloodsample has a volume of between 60 mL and no more than 250 mL. The firstvessel is for storing the target cell-poor fraction separated from theblood sample. The second vessel is for storing the target cell-richfraction separated from the blood sample. The first valve is upstream ofthe first vessel and the second vessel, and downstream of the bloodseparation component. The second valve is for providing selective fluidcommunication between the first vessel and the second vessel. Theconcentration and flow logic and control component is configured to:control the first valve to remove the target cell-poor fraction to thefirst vessel and to remove the target cell-rich fraction to the secondvessel; determine a first volume of the target cell-poor fraction in thefirst vessel to mix with the target cell-rich fraction in the secondvessel in order to form a target cell-rich concentrate having aconcentration of target cells that is within a target concentrationrange for studying or defining a dose-response relationship in apatient; determine whether the target cell-rich concentrate has aconcentration of target cells within the target concentration range; andcontrol the second valve to transfer the first volume of the targetcell-rich fraction from the first vessel to the second vessel to formthe target cell-rich concentrate. The target concentration range isbetween 1.0 and 1.5×106 target cells/μL, and the cell concentratingsystem is autologous.

In another aspect, a method of generating a target cell-rich concentrateis disclosed. The method includes obtaining a target blood sample havinga volume of between 60 mL and 250 mL, and inputting the target bloodsample into a cell concentration system. The method also includescausing the cell concentration system to: separate the target bloodsample into a red blood cell fraction, a target cell-poor fraction, anda target cell-rich fraction; remove the target cell-poor fraction to afirst vessel; remove the target cell-rich fraction to a second vessel inselective fluid communication with the first vessel; determine a firstvolume of the target cell-poor fraction in the first vessel to mix withthe target cell-rich fraction in the second vessel in order to form atarget cell-rich concentrate having a concentration of target cells thatis within a target concentration range for studying or defining adose-response relationship in a patient; determine whether the targetcell-rich concentrate has a concentration of target cells within thetarget concentration range; and transfer the first volume of the targetcell-rich fraction from the first vessel to the second vessel to formthe target cell-rich concentrate. The target concentration range isbetween 1.0 and 1.5×106 target cells/μL. The target cell-richconcentrate is an autologous target cell-rich concentrate of the targetblood sample.

In another aspect, a method of autologous therapeutics is disclosed. Themethod includes: extracting a target blood sample from a patient, thetarget blood sample having a volume of between 60 mL and 250 mL, andinputting the target blood sample into a cell concentration system. Themethod also includes causing the cell concentration system to: separatethe target blood sample into a red blood cell fraction, a targetcell-poor fraction, and a target cell-rich fraction; remove the targetcell-poor fraction to a first vessel; remove the target cell-richfraction to a second vessel in selective fluid communication with thefirst vessel; determine a first volume of the target cell-poor fractionin the first vessel to mix with the target cell-rich fraction in thesecond vessel in order to form a target cell-rich concentrate having aconcentration of target cells that is within a target concentrationrange for studying or defining a dose-response relationship in apatient; determine whether the target cell-rich concentrate has aconcentration of target cells within the target concentration range; andtransfer the first volume of the target cell-rich fraction from thefirst vessel to the second vessel to form the target cell-richconcentrate. The target concentration range is between 1.0 and 1.5×106target cells/μL. The target cell-rich concentrate is an autologoustarget cell-rich concentrate of the target blood sample. The method alsoincludes injecting the autologous target cell-rich concentrate into thepatient.

BRIEF DESCRIPTION OF THE DRAWINGS

Various objects and advantages and a more complete understanding of thepresent invention are apparent and more readily appreciated by referringto the following detailed description and to the appended claims whentaken in conjunction with the accompanying drawings:

FIG. 1 illustrates a flow diagram of operations and components of asystem for autologous infusion of platelet-rich plasma concentrates orbone marrow cell concentrates having specified concentration ranges.

FIG. 2 illustrates a method for generating a PRP concentrate of aspecific platelet concentration.

FIG. 3 illustrates another method for generating a PRP concentrate of aspecific platelet concentration.

FIG. 4 illustrates yet another method for generating a PRP concentrateof a specific platelet concentration.

FIG. 5 illustrates a method for generating a BMC-rich concentrate of aspecific BMC concentration.

FIG. 6 illustrates another method for generating a BMC concentrate of aspecific BMC concentration.

FIG. 7 illustrates yet another method for generating a BMC concentrateof a specific BMC concentration.

FIG. 8 illustrates a block diagram representation of another system forgenerating a target concentration and/or volume of platelets or bonemarrow cells (BMCs).

FIG. 9 illustrates a PRP or BMC concentrating apparatus.

FIG. 10 illustrates one embodiment of a package comprising thosecomponents of the apparatus that can be disposable.

FIG. 11 illustrates how a syringe can be used to provide a whole bloodor bone marrow sample to the apparatus of FIG. 9.

FIG. 12 illustrates a user interacting with the apparatus of FIG. 9, forinstance, via a touchscreen embodiment of the user interface.

FIG. 13 illustrates how a syringe can be used to remove contents of thefirst vessel illustrated in FIGS. 9-13.

FIG. 14 shows a diagrammatic representation of one embodiment of amachine in the exemplary form of a computer system within which a set ofinstructions can execute for causing a device to perform or execute anyone or more of the aspects and/or methodologies of the presentdisclosure.

DETAILED DESCRIPTION

There is a long-felt need in the art for systems, methods, and apparatuscapable of generating narrow concentration ranges of PRP and BMCC. Withsuch well-known concentrations, clinical studies of the effects ofdifferent concentrations on patients will be greatly improved.

For the purposes of this disclosure, bone marrow cells (BMCs) and bonemarrow cell concentrates (BMCCs) include, but are not limited to, anyone or more of the following: stem-like cells (pluripotent cells) (e.g.,monocytes), white blood cells, platelets, neutrophils, lymphocytes,eosinophils, and basophils. In fact, BMCs include any cells found in abone marrow sample and a BMCC includes any of the cells found in a bonemarrow sample, but at higher-than-natural concentrations. Throughoutthis disclosure, a BMC-rich fraction refers to a substance or fluidhaving an increased concentration of any one or more target or desiredcells as compared to the natural concentration in a human body. ABMC-poor fraction refers to a substance or fluid having a decreasedconcentration of any one or more target or desired cells as compared tothe natural concentration in a human body.

The concentrates of this disclosure can comprise one or more of cells,signals, and scaffolds. These components can be harvested and generatedfrom many sources. For instance, in an autologous system, the patient istreated using components removed from the same patient.

For the purposes of this disclosure, “cells” include mesenchymal stemcells (MSCs) originating from bone marrow, fat, blood, synovium, orother tissues. Cells also include pluripotent cells from bone marrow,blood, and other sources. Cells further include native tissue cells,which can be stimulated to grow and proliferate via signals.

For the purposes of this disclosure, “signals” refer to human growthfactor proteins, and can be derived from platelets or from autocrine(cell-cell) sources. Additionally, for the purposes of this disclosure,“scaffolds” refer to a mechanical matrix made from a blood-based fibrinthat can be used to deliver and provide a platform for tissue growth invivo. As herein disclosed, there is often a blood based fibrin thatcreates a scaffold by inducing a clotting cascade in the blood, convertsfibrinogen to fibrin, and creates a mechanical fibrin matrix. Cellsand/or growth factor proteins can be implanted or ‘seeded’ into ascaffold where the scaffold supports three-dimensional tissue formationfrom the seed.

Whole blood is human blood from a standard blood donation. Whole bloodcan be combined with an anticoagulant during a collection process, butis generally otherwise unprocessed. The capitalized “Whole Blood” meansa specific standardized product for transfusion or further processing.The lower case “whole blood” encompasses any unmodified collected blood.

Flow cytometry (FCM) is a technique for counting and examiningmicroscopic particles, such as cells and chromosomes, by suspending themin a stream of fluid and passing them by an electronic detectionapparatus. It allows simultaneous multiparametric analysis of thephysical and/or chemical characteristics of up to thousands of particlesper second.

For the purposes of this disclosure, separation means chemicallyseparating components, but not necessarily physical separation. Forinstance, centrifugation separates a fluid into layers that areprimarily isolated from each other based on particle mass. However,there may be some overlap between the layers, and such is included inthis disclosure's use of the term separation. At the same time, if thoselayers were then split into different vessels, this would also beconsidered separation.

FIG. 1 illustrates a flow diagram of operations and components of asystem for autologous infusion of platelet-rich plasma concentrates orbone marrow cell concentrates having specified concentration ranges.FIG. 1 will be described in conjunction with FIGS. 2-4, which describemethods of the same. A whole blood sample or bone marrow cell (BMC)sample 102 is taken from a patient 104 (Blocks 202, 302, 402, 502, 602,702). The sample 102 enters a red blood cell (RBC) separation component106 (first separation component), which separates RBCs from a plasmafraction of the sample 102 (Blocks 204, 304, 404, 504, 604, 704). TheRBCs can be stored in an RBC storage or disposal vessel 108 (Blocks 206,306, 406, 506, 606, 706).

The plasma fraction can then be separated in a PRP or BMC fractionseparation component 110 (second separation component). The secondseparation component 110 separates the plasma fraction into aplatelet-poor plasma (PPP) fraction and a platelet-rich plasma (PRP)fraction, or a BMC-poor fraction and a BMC-rich fraction (Blocks 210,310, 410, 510, 610, 710). The PPP or BMC-poor fraction can be stored ina PPP or BMC-poor storage or disposal vessel 112.

The PRP or BMC-rich fraction can be mixed with an aliquot of the PPP orBMC-poor fraction in a mix component 114 (Blocks 212, 312, 412, 512,612, 712). Alternatively, rather than removing the entire PPP orBMC-poor fraction and then remixing an aliquot of the removed fraction,a first aliquot of the PPP or BMC-poor fraction can be removed to thePPP or BMC-poor storage or disposal vessel 112, while a second aliquotis left with the PRP or BMC-rich fraction.

The mix component 114 can be controlled by a concentration and flowlogic and control 116. The concentration and flow logic and control 116determines a volume of the aliquot to be added to the PRP or BMC-richfraction based on a determination of the total number of platelets orBMCs that were in the whole blood sample or BMC sample 102 (Blocks 208,308, 408, 508, 608, 708). Such determination can be made by measuring aplatelet or BMC concentration 120, 122, 124, at various points in theprocess via a platelet or BMC concentration measurement component 118.For instance, concentration 120 can be measured from the whole blood orBMC sample (Blocks 408, 708). Concentration 122 can be measured afterthe RBC separation, thus measuring the platelet or BMC concentration ina plasma (Blocks 208, 508). As another alternative, a concentration 124can be measured after the PRP or BMC-rich fraction has been separated insecond separation component 110 (Blocks 308, 608). This concentration124 is measured from the PRP or BMC-rich fraction. All threeconcentrations 120, 122, 124 should be identical measurements, althoughthey may vary since the separations are not ideal and thus someplatelets or BMC may end up in the RBC fraction or the PPP or BMC-poorfraction. Since platelets and bone marrow cells can separate, anagitation step may be added before any of the first, second, or thirdconcentrations 120, 122, 124 are measured.

The concentrations 120, 122, 124 can be multiplied by the sample 102volume, the plasma volume, or the PRP or BMC-rich fraction volume,respectively, to determine a total number of platelets or BMCs in thePRP or BMC-rich fraction. The concentration and flow logic and control116 divides this total number by a target concentration (e.g.,0.8-2.0×10⁶ platelets/μL or 1.0-1.5×10⁶ platelets/μL) to get a totaltarget volume that the mix should attain. The amount of PPP or BMC-poorfraction to mix with the PRP or BMC-rich fraction is the differencebetween the total target volume and the volume of the PRP or BMC-richfraction (for a further explanation, see Equations (1)-(6)).

The mixture of the aliquot of PPP or the aliquot of the BMC-poorfraction and the PRP or BMC-rich fraction can be stored in a PRP orBMC-rich concentrate storage vessel 126. This mixture can also bereferred to as an PRP or BMC-rich concentrate and can have a targetconcentration of platelets or BMCs and/or a target volume. The PRP orBMC-rich concentrate is then available for infusion back into thepatient 104 (Blocks 216, 316, 416, 516, 616, 716). The PRP or BMCconcentrate can be compatible with blood bank cross-matched blood.

In one alternative, an optional anticoagulant 128 can be added to thewhole blood sample or BMC sample 102 before the first separation inorder to help prevent the platelets from activating (Blocks 222, 322,422, 522, 622, 722). Similarly, a solution 130 to reverse theanticoagulant 128 can be added to the PRP or BMC-rich concentrate beforeor after the concentrate has reached the PRP or BMC-rich concentratestorage vessel 126.

Another embodiment can optionally agitate the concentrate before orafter the concentrate reaches the PRP or BMC-rich storage vessel 126 viaan agitation component 132. This agitation can help mix the concentrateand/or begin activation of the platelets in the concentrate. Theagitation component 132 can be separate from or integrated with the PRPor BMC-rich concentrate storage vessel 126. Agitation can involvestirring in one embodiment.

A user interface 134 can be used to interface with, control, and monitorthe process via the concentration and flow logic and control 116. Theuser interface 134 can enable a user (e.g., a doctor, nurse, ortechnician) to monitor parameters of the procedure as well as to provideinputs such as a target concentration and/or a target volume of the PRPor BMC-rich concentrate.

In one embodiment, another measurement and analysis of the PRP or BMCconcentration of the concentrate is performed before infusion into thepatient 104 (Blocks 214, 314, 414, 514, 614, 714). If the concentratefalls within a target range of concentrations, then the concentrate canbe provided to the patient 104 (Blocks 216, 316, 416, 516, 616, 716).However, if the concentration does not fall within the target range,then the concentrate can be remixed with the RBC fraction (Blocks 218,318, 418, 518, 618, 718) and passed back through the process startingwith the RBC separation (Blocks 204, 304, 404, 504, 604, 704), until thePRP or BMC-rich concentrate falls within the target range. Where thedetermination of platelet or BMC concentration 120 was made from thesample 102 (Blocks 408, 708), there may be an optional determination oftotal platelets (Block 424) or total number of BMCs (Block 724) beforethe first separation (Blocks 404, 704).

In an embodiment an optional anticoagulant 128, such as ACDA, can beadded to the whole blood or BMC sample 102 before any separatingprocedures begin. Since the separation procedure as well as the meremovement of the platelets between vessels, centrifuges, or any othercomponents of the system entail agitation of the platelets, andagitation can initiate undesired activation (clotting) of the platelets,the anticoagulant 128 helps to preserve the platelets in a non-activatedstate until they are ready to be infused back into the patient 104. Inone embodiment, 3 mL of anticoagulant can be added to 50 mL of wholeblood or 5 mL of anticoagulant added to 50 mL of BMC. In the case of aBMC sample 102, an aspiration syringe can be flushed before the BMCsample 102 is passed to the separation component 106. For instance, anaspiration syringe can be flushed with heparin (e.g., 1,000 U/mL).

In one embodiment, a whole blood or BMC sample 102 is between 60-250 mL,while in another the sample 102 is between 60-120 mL. The Whole Bloodcan be sourced from an intravenous catheter. The BMC can be harvested byneedle aspiration from an intramedullary cavity of the anterior orposterior hip, shoulder, or knee, although other methods and sources foracquiring BMC are also envisioned.

While the presently discussed systems and methods describe autologoussystems and methods (infusion back into the source patient 104), inother embodiments, the source patient and the patient to be infused canbe different.

A BMC sample 102 may be put through an initial removal stage forremoving high molecular weight components such as bone particulates. TheBMC sample 102 can then be filtered to remove any remaining fat and/orlarge particles (Blocks 526, 626, 726). A 170-260 μm filter can be usedin one instance.

The red blood cell (RBC) separation component 106 separates the sample102 into an RBC fraction and a non-RBC fraction or plasma fraction. Inthe case of a whole blood sample 102, the non-RBC fraction may includenucleated or white blood cells (WBCs), platelets, and serum. In the caseof a BMC sample 102, the non-RBC fraction can include plasma, platelets,and WBCs (including pluripotent cells).

The RBC separation component 106 can carry out a ‘soft spin’ viacentrifuge in one embodiment. In one embodiment, the soft spin caninvolve centrifugation at 2500-3000 RPM for 8-15 minutes where wholeblood is involved, and 2400 RPM for 10 minutes where BMCs are thesource, although these exemplary specifications are not limiting. Incentrifugation, the plasma fraction is the fraction of the sample 102that accumulates above the RBC fraction or closer to a center of thecentrifuge (RBCs are the heaviest components of an RBC sample). The RBCsaccumulate below the plasma fraction since they tend to be heavier.Within the plasma fraction, the centrifugation may also cause furtherseparation between mainly plasma particles and a “buffy coat,” which mayinclude nucleated cells, platelets, plasma, and WBCs. The buffy coattends to be found between the plasma and the RBC fraction.

The RBC separation component 106 can alternatively use a variety ofother separating components and methods including, but not limited to,separation according to microfluidic channel separation, polymer-basedseparation, acoustophoresis, various lab-on-chip or lab-on-CD (compactdisc) technologies, flow cytometry, dielectrophoresis, laser impedance,flow cytometry, and use of fluorescent or other markers. Microfluidicchannel separation involves passing a fluid through channels of varyingdiameters such that particles of varying sizes can only fit throughcertain channels, and thus particles can be separated depending on whichchannels they are able to pass through. Acoustophoresis involves the useof acoustic signals to separate components of a fluid. The polymer-basedmethod involves adding a polymer to the plasma fraction that causesplatelets to separate from the plasma. The RBC separation component 106should be selected so as to minimize platelet activation and maximizeplatelet yield.

The RBC fraction can be directed to an RBC storage vessel 108 for latertransfusion or disposal via a valve or pump. The plasma fraction can bedirected to a different vessel or a portion of the system where furtherseparation is to occur. In some embodiments, the RBC fraction can remainin the first separation component 106 after the plasma fraction has beenremoved, and since the first separation component 106 may be disposable,the RBC fraction can be left in the first separation component 106 fordisposal. In such an embodiment, a separate RBC storage or disposalvessel 108 is not implemented.

In the case of a plasma fraction derived from bone marrow, a filtrationprocess can be used to further filter the plasma (e.g., a ˜200 μm meshfilter). For instance, any anti-coagulated flushed syringe can be usedto pull the plasma fraction through a filter.

Once the RBC fraction is separated from the plasma fraction, the plasmacan pass to the plasma-rich platelet (PRP) or the BMC-rich fractionseparation component 110 (the second separation component). The secondseparation component 110 separates the plasma into a platelet-poorplasma (PPP) fraction and a platelet-rich plasma (PRP) fraction or aBMC-poor fraction and a BMC-rich fraction. In many cases, the PPPfraction or the BMC-poor fraction are the larger fractions. The PPPfraction tends to have a low or negligible concentration of platelets.

In some embodiments, the second separation component 110 is the RBCseparation component 106 (also known as a first separation component)and will hereinafter be referred to as the separation component 106/110.For instance, a single centrifuge can be used to separate the sample 102into an RBC fraction, a PRP or BMC-rich fraction, and a PPP or BMC-poorfraction. This can involve first removing the plasma fraction from theseparation component 106/110 and then passing the plasma fraction backinto the separation component 106/110 or leaving the plasma fraction inthe separation component 106/110 while the RBC fraction is firstseparated and removed, and then the plasma fraction is separated.Alternatively, the three fractions can be simultaneously separated.

In other embodiments, the first separation component 106 and the secondseparation component 110 are separate and different components. Forinstance, two centrifuges may be used. However, the first and secondseparation components 106, 110 can also be different types ofcomponents. In one case, a centrifuge can be used as the firstseparation component 106 and a set of microfluidic pores of differentdiameter can be used as the second separation component 110. Many othervariations are also possible.

Where the second separation component 110 is a centrifuge, or where bothseparation components 106, 110 are the same centrifuge, a second or‘hard spin’ can be performed on the plasma fraction. The hard spin caninvolve a 2800-3200 RPM spin for 5-8 minutes where whole blood is thesource and 3400 RPM for 6 minutes where BMCs are the source, althoughthese exemplary parameters are not limiting. The hard spin separates theplasma into a PPP fraction and a PRP fraction, or a BMC-poor fractionand a BMC-rich fraction, where the PPP fraction or the BMC-poor fractiontends to be a larger upper layer comprising lighter particles and only asmall concentration of platelets or BMCs. Underneath this layer is asmaller “buffy coat” or “pellet” comprising the heavier platelets orBMCs, which accumulate towards an outer radius of the centrifuge.

The PRP or BMC-rich fraction separation component 106 can alternativelyuse a variety of other separating components and methods including, butnot limited to, separation according to microfluidic channel separation,polymer-based separation, acoustophoresis, various lab-on-chip orlab-on-CD (compact disc) technologies, flow cytometry,dielectrophoresis, laser impedance, flow cytometry, and use offluorescent or other markers.

Once the plasma fraction is separated, the PPP or BMC-poor fraction canbe directed to a PPP or BMC-poor storage or disposal vessel 112. Forinstance, one or more valves and pumps can be used to direct the fluidflow.

In one embodiment, rather than remove the PPP or BMC-poor fraction, onlyan aliquot of this fraction is removed to the storage or disposal vessel112. Selection of the volume of this aliquot will be discussed laterrelative to the concentration and flow logic and control 116 and the mixcomponent 114.

The mix component 114 can agitate the mixture of PRP and PPP or BMC-richand BMC-poor in order to suspend cells within the fluid. Alternatively,the mere action of forcing the two substances into the same vessel canachieve satisfactory mixing.

The volume of the aliquot of PPP or BMC-poor plasma fraction to mix withthe PRP or BMC-rich plasma fraction in order to achieve the targetconcentration is determined as follows. For readability, thisdescription will refer only to platelets, but is equally applicable toBMCs. First a total number of platelets, P_(total), in the sample 102 isdetermined. This is done by: (a) measuring or estimating a plateletconcentration, PC₀, (e.g., platelet concentrations 120, 122, 124) and(b) multiplying the platelet concentration, PC₀, by a volume, V₀, of thefluid in which the platelet concentration was measured in. This is shownin Equation (1) as follows:

P _(total) =PC ₀ ×V ₀   (1)

There are at least three locations or times during the process where theconcentration PC₀ and the volume V₀ can be determined. First, the firstconcentration 120 can be measured from the whole blood sample 102.Second, the second concentration 122 can be measured after the firstseparation component 106 has separated the sample 102 into a RBCfraction and a plasma fraction. The second concentration 122 can bemeasured either before or after the RBC fraction has been moved to theRBC storage or disposal vessel 108. Either way, the second concentration122 is taken from the plasma fraction not from the RBC fraction. Third,the third concentration 124 can be measured after the plasma fraction isseparated into an RBC fraction and a PPP fraction.

The concentration PC₀ is passed to the concentration and flow logic andcontrol 116 where it is used to determine a total number of platelets inthe volume V₀ of fluid that was measured. The platelet or BMCconcentration measurement component 118 can also measure the volume V₀(e.g., via a flow meter) in which the concentration PC₀ was measured.Alternatively, the concentration PC₀ can be measured within a spacehaving a known volume V₀. For instance, the sample 102, or the plasmafraction, or the PRP can have known volumes. In another embodiment, auser (e.g., a doctor, nurse, or technician) can enter the volume V₀ intothe user interface 134, which provides the volume V₀ to theconcentration and flow logic and control 116. Thus, the concentrationand flow logic and control 116 can utilize both the concentration PC₀and the volume V₀ of the fluid measured to solve for the total number ofplatelets PTOTAL according to Equation (1).

To achieve a target (e.g., user-defined) PRP concentration, PC_(t), somealiquot of the PPP fraction is mixed with the PRP fraction. An exemplaryrange of target PRP concentrations, PC_(t), is 0.8-2.0×10⁶ platelets/μLor 1.0-1.5×10⁶ platelets/μL. An exemplary target PRP concentration,PC_(t), is 1.5×10⁶ platelets/μL. In one embodiment this involvesremoving some of the PPP fraction then mixing the remaining PPP fractionand the PRP fraction. In another embodiment, the PPP fraction isremoved, and then a portion of the PPP fraction is mixed back in withthe PRP fraction. In both cases, knowing how much of the PPP fraction tomix with the PRP fraction can be determined by calculating a targetvolume for the mixture V_(T). This value is given as follows:

$\begin{matrix}{V_{t} = \frac{P_{total}}{{PC}_{t}}} & (2)\end{matrix}$

The target volume, V_(t), is equal to the number of platelets,P_(total), divided by the target PRP concentration PG. Equation (2) canbe simplified by substituting Equation (1) for P_(total) in Equation (2)as follows:

$\begin{matrix}{V_{t} = \frac{{PC}_{0} \times V_{0}}{{PC}_{t}}} & (3)\end{matrix}$

To achieve the target PRP concentration PC_(t) an aliquot V_(PPP) of thePPP fraction is mixed with the PRP fraction, having a volume V_(PRP)(measured by a flow meter, for example), so that the combination equalsthe target volume V_(t). This can be written as Equation (4) and solvedfor the aliquot V_(PPP) in Equations (5) and (6) as follows:

$\begin{matrix}{V_{t} = {V_{PPP} + V_{PRP}}} & (4) \\{{V_{PPP} + V_{PRP}} = \frac{{PC}_{0} \times V_{0}}{{PC}_{t}}} & (5) \\{V_{PPP} = {\frac{{PC}_{0} \times V_{0}}{{PC}_{t}} - V_{PRP}}} & (6)\end{matrix}$

Thus, where a portion of the PPP fraction is removed, some of the PPPfraction is removed until the volume of the remaining PPP fraction andthe PRP fraction equals V_(t). Said another way, a portion of the PPPfraction is removed until the remaining PPP fraction has a volume equalto V_(PPP) as in Equation (6). Where the entire PPP fraction is removed,and then an aliquot having volume V_(PPP) is added back with the PRPfraction, V_(PRP), the aliquot of the PPP fraction can be selected sothat the combined volume of the aliquot of the PPP fraction, V_(PPP),and the volume of the PRP fraction, V_(PRP), equals the target volumeV_(t).

Equation (6) may suffer from the fact that some platelets are removedwith the RBC by the first separation component 106, and with the PPPfraction by the second separation component 110, however such numberscan be considered negligible when the separations are carried out withcare.

As noted earlier, the derivation of Equation (6) was described relativeto PRP, but is equally applicable to BMC-rich plasma. In particular,Equation (7) shows Equation (6) as applied where bone marrow is thesource and a BMC-rich plasma concentrate is the end goal.

$\begin{matrix}{V_{{BMC} -} = {\frac{{BMCC}_{0} \times V_{0}}{{BMCC}_{t}} - V_{{BMC} +}}} & (7)\end{matrix}$

The concentration of the BMCs, BMCC₀, can be measured from the BMCsample 102, after the first separation component 106 has separated theBMC sample 102 into an RBC fraction and a plasma fraction, or after thesecond separation component 110 has separated the plasma fraction into aBMC-poor fraction and a BMC-rich fraction. The target concentration ofBMCs is BMCC_(t). The volume of the BMC-rich fraction is V_(BMC+), andthe volume of the aliquot of the BMC-poor fraction to be mixed with theBMC-rich fraction is V_(BMC). Once again, the aliquot of the BMC-poorfraction, V_(BMC−), can be added to the BMC-rich fraction or left withthe BMC-rich fraction as the rest of the BMC-poor fraction is removed.Equations (6) and (7) can also be adapted for use with any target bloodcells such as white blood cells (e.g., monocytes), platelets, bonemarrow cells, and “stem-like” or “pluripotent” cells.

During this process, the concentration and flow logic and control 116can provide instructions to a human user via the user interface 134indicating how much of the PPP or BMC-poor fraction to mix with the PRPor BMC-rich fraction. Alternatively, the concentration and flow logicand control 116 can provide a value for the target volume V_(t) to auser via the user interface 134. In another embodiment, theconcentration and flow logic and control 116 can automatically controlthe mix component 114 and control the volume of the PPP or BMC-poorfraction that is mixed with the PRP or BMC-rich fraction. For instance,the concentration and flow logic and control 116 can control a valve andpump that either removes a certain amount of the PPP fraction or adds acertain amount of the PPP fraction back in with the PRP or BMC-richfraction.

The user interface 134 can display information describing themeasurements that the platelet or BMC concentration measurementcomponent 118 is performing. The user interface 134 can be part of orseparate from the concentration and flow logic and control 116. Thisinformation can also be stored in a memory or transferred throughtelemetry to a central record keeping system.

The user interface 134 can also be used by a user (e.g., doctor, nurse,or technician) to set a target PRP or BMC concentration PC_(t) orBMCC_(t) and a target volume V_(t). The user interface 134 can also beused to request and display results from a concentration analysis of thePRP or BMC-rich concentrate. Such analysis can be performed after theconcentrate has been formed, but before the concentrate is administeredto a patient, to ensure that the desired concentration was created.

The platelet or BMC concentration measurement component 118 can includea variety of hemoanalysis machines and methods. For instance,fluorescent activated cell sorting (FACS) can determine theconcentration of a specific pluripotent, stem-like cell, within a fluidbased on antibody cell surface markers. Other exemplary embodiments ofthe concentration measurement component 118 include those for opticalmicroscopy, optical light scattering, and electrical impedance. Opticalmicroscopy involves a computer-controlled pattern and shape recognitioncomponent and logic that counts and differentiates particles by shapeand size. In some instances, this method cannot be performedcontinuously, and may therefore require sampling of discrete portions ofa fluid. In one embodiment, sampling of a thin fluid layer may beimplemented. Optical light scattering may use a hydrodynamic focusedstream of fluid and the method can count different types of cells andmolecules especially where fluorescence markers or anti-bodies are used.Electrical impedance may use a hydrodynamic focused steam of fluid.

In some embodiments, either of these concentration measurementcomponents 118 can be combined with a particle separation component. Forinstance, a microfluidic channel device could be used to separateparticles within a PRP fraction, while a light scattering devicemeasures a number of particles in each fluid stream. The combination ofa particle separation device and a particle counting device can bebeneficial where the particle counting device is not able to distinguishbetween different types of cells or particles within the same stream.

In one embodiment, hemoanalysis can be performed on the PRP or BMC-richconcentrate (Blocks 214, 314, 414, 514, 614, 714). This hemoanlaysis canbe in addition to or in the alternative to one or more previoushemoanalysis steps. For instance, in one embodiment, hemoanalysis can beperformed on the whole blood sample or BMC sample and on theconcentrate. In another embodiment, hemoanalysis can be performed afterthe first separation as well as on the concentrate.

In an embodiment, autologous thrombin or a fibrin matrix can be preparedfrom some or all of the PPP or BMC-poor fraction in the PPP or BMC-poorstorage or disposal vessel 112 (Blocks 220, 320, 420, 520, 620, 720).The effects of the anticoagulant 128 can be reversed, for example, byaddition of CaCl (e.g., a 10% solution of CaCl can be added to the PPPor BMC-poor fraction). The PPP or BMC-poor fraction and whateversubstance is used to reverse the anticoagulant 128 can be agitated(e.g., for approximately 1 minute) resulting in formation of a fibrinclot. During or after clot formation, additional PPP or some of theBMC-poor fraction can be added to the mixture. Further agitation can beperformed and the mixture can be given time for the clot to continue toform. When clotting is complete, the clot can be removed and manuallycompressed (or re-centrifuged to compress).

In the case of autologous thrombin formation, prothrombin protein iscleaved in the process of reversing the anticoagulant and thrombin isproduced. Removal of the clot leaves serum and a low concentration ofthrombin. The thrombin can be combined with implanted PRP to initiateand regulate a release of platelet growth factors to stimulateregenerative responses.

Where fibrinogen in PPP is activated by calcium reversal of theanticoagulant or the addition of the autologous thrombin to inducecleavage of the fibrinogen to fibrin, a fibrin matrix or scaffold isformed. The matrix or scaffold can be implanted to act as a scaffoldfrom which native or implanted cells can attach and proliferate to formnew tissue.

The clot can be transferred to an implantation destination in thepatient 104. The implantation destination can be a joint or area whereregeneration is desired, or any other selected site in the patient 104.After the clot is removed, the remaining PPP or BMC-poor can be used toimprove platelet activation by combination with the PRP or BMC-richconcentrate either in-vitro (creating a PRP membrane) or in-vivo(creating an activated PRP). Although the thrombin or fibrin matrix canbe autologously applied, they can also be implanted into another patientother than the patient 104.

The autologous thrombin or fibrin matrix can be removed via syringe andimplanted into the patient 104. In one alternative, a fibrin matrix orAutologous Platelet Gel (APG) can be autonomously created from activatedPPP and a portion of the PRP concentrate. Other products includeactivated PPP containing autologous thrombin. The manual or autonomousprocedures can be carried out in the operating room or desired treatmentlocation.

PPP can include an acellular blood fraction comprising ˜55% of bloodvolume, 91% water content, and residual proteins. Although PPP ispreferably devoid of platelets, in practice a small residual plateletfraction may be observed.

In some embodiments, the platelet or BMC concentration measurementcomponent 118 can also measure other blood component concentrations suchas white blood cell concentration. Other concentrations that can bemeasured include those for red blood cells, Neutrophils, Lymphocytes,Monocytes, Eosinophils, and Basophils. These alternative measurementscan be used in embodiments, where concentrations of non-platelet andnon-BMC blood cells are also being concentrated to a targetconcentration or target concentration range.

Sometimes the mixing of mix component 114 is not sufficient to suspendthe platelets in the PRP or BMC concentrate. In these cases, or toenhance platelet activation, an optional agitation component 132 canagitate the PRP or BMC concentrate. Where BMCs are used, the pellet canbe reconstituted (increased liquid content) in one of three mediums toease infusion into the patient 104: accellular bone marrow aspirate;blood plasma; or PRP.

One or more of the first and second separation components 108, 110, theplatelet or BMC concentration measurement component 118, theconcentration and flow logic and control 116, and the mix component 114can be discarded after use in order to enhance sterility within thesystem 300. All storage vessels 108, 112, 126 can also be disposable.

The system 100 can take a variety of forms including a miniaturized formsuch as a handheld or bench top system or other portableimplementations. Portability can include being handheld, lightweight,and/or supported by a cart. The system 100 could also be designed sothat continuous or intermittent infusions of PRP could be administeredto a patient over a period of time. In an embodiment, the system 100 canbe implemented wholly or partially as one or more microelectromechanicalsystems (MEMS) devices and/or as a lab on a chip.

The systems and methods herein disclosed can also achieve a number offurther goals via various alternative embodiments. In an embodiment,blood product sterility is maintained, for instance via inclusion of oneor more disposable components. In an embodiment, blood is collected froma patient prior to an operation and used to prepare a final PRP productfor the operation. In an embodiment, a PRP membrane is created.Alternatively, activated PRP is used to create PRP within an autologousfibrin matrix. In another embodiment, the system 100 can create a PRP orBMC concentrate within 30 minutes.

The systems and methods can incorporate, or be incorporated into,existing technologies and components used in automated transfusion andhemoanalysis machines. In addition to counting, measuring and analyzingred blood cells, white blood cells, platelets, or other bloodcomponents, automated hematology analyzers can also measure the amountof hemoglobin or chemical regulators in the blood and within each redblood cell.

The systems and methods described herein can be applied to injured orpathologic tissue to stimulate and/or enhance repair or regeneration.Methods of implantation can include percutaneous (injection) orintra-operative (surgical) application. Other embodiments can include animplanted or partially implanted continuous or periodic delivery systemfor providing defined dosages over time.

Other sample sources that can be used in place of whole blood and bonemarrow including fat, synovium, and other tissue.

FIG. 2 illustrates a method for generating a PRP concentrate of aspecific platelet concentration. The method 200 includes obtaining awhole blood sample in obtain sample operation 202 and optionally addingan anticoagulant in add anticoagulant operation 222. Then a firstseparation operation 204 separates the sample into a red blood cell(RBC) fraction and a plasma fraction. The RBC fraction can be discardedor re-perfused into a patient in a discard or re-perfuse operation 206.

The total number of platelets, P_(total), in the plasma fraction can bedetermined in determine operation 208. This operation 208 utilizes atleast one measurement such as a concentration measured by a hemoanalysisapparatus. The platelet concentration, PC₀, can be multiplied by avolume V₀ of the liquid, as measured by a flow meter for instance, fromwhich the concentration measurement was made. The volume, V₀, times theconcentration, PC₀, gives a total number of platelets, P_(total). Theplasma fraction is then separated further via second separationoperation 210 (e.g., microfluidic channel separation or centrifugation),in which a platelet-rich plasma (PRP) fraction (high concentration ofplatelets) and a platelet-poor plasma (PPP) fraction (negligibleplatelet concentration) are generated.

The PRP fraction and an aliquot of the PPP fraction, V_(PPP), are mixedin mix operation 212. The volume of the aliquot, V_(PPP), can be basedon a target platelet concentration, PC_(t), and the total number ofplatelets, P_(total) (e.g., Equation (6)). The mix operation 212 forms aPRP concentrate which can then be provided to a patient in provide PRPconcentrate to patient operation 216. The remaining PPP fraction canalso be used to form an autologous thrombin preparation for implantationinto a patient in an autologous thrombin preparation operation 220.

Optionally, after the PRP concentrate is formed in mix operation 212,the platelet concentration can be checked to ensure that the plateletconcentration falls within a target range (or within a margin of errorof a target platelet concentration) in an optional decision 214. If thedecision 214 finds that the PRP concentrate falls within the targetrange or within a margin of error of the target concentration, then thePRP concentrate can be provided to a patient. If not, then theconcentrate can be remixed with the RBC fraction and the process can berepeated starting with the first separation operation 204.

Since settling may cause the concentrate to separate at least partiallyinto a PPP layer and a PRP layer, an agitation operation may optionallybe used to suspend the platelets prior to infusion.

FIG. 3 illustrates another method for generating a PRP concentrate of aspecific platelet concentration. The method 300 is nearly identical tothe method 200 with the exception that the determine operation 308 takesplace on the PRP fraction after the second separation operation 310,rather than between the first and second separation operations 304, 310.In some embodiments, the first and second separation operations 304, 310can be performed in a single operation by a single separation component(e.g., a centrifuge that creates an RBC fraction, a PRP fraction, and aPPP fraction).

FIG. 4 illustrates yet another method for generating a PRP concentrateof a specific platelet concentration. The method 400 is nearly identicalto the methods 200 and 300 with the primary exception that the determineoperation 408 takes place on the whole blood sample, before either ofthe separation operations 404, 410.

Another distinction from the methods 200 and 300 is that the determinetotal number of platelets in whole blood sample based on at least onemeasurement operation 408 can be carried out in parallel with anoptional add anticoagulant to whole blood sample operation 422.Alternatively, both operations 408, 422 can be carried out atoverlapping or non-overlapping times between an obtain whole bloodsample operation 402 and the first separation operation 404.

A final distinction is that after an optional remix red blood cellfraction with PRP concentrate operation 418, the method 400 may includean optional determine total number of platelets operation 424. Thisoperation 424 can determine a total number of platelets in the mixtureof RBCs and the PRP concentrate after they have been remixed, in theevent that the PRP concentrate does not fall within a targetconcentration range according to a decision 414. In some embodiments,the first and second separation operations 404, 410 can be performed ina single operation by a single separation component (e.g., a centrifugethat creates an RBC fraction, a PRP fraction, and a PPP fraction).

FIG. 5 illustrates a method for generating a BMC-rich concentrate of aspecific BMC concentration. The method 500 includes obtaining a BMCsample in obtain sample operation 502 and optionally adding ananticoagulant in add anticoagulant operation 522. Then a firstseparation operation 504 separates the sample into a red blood cell(RBC) fraction and a plasma fraction. The RBC fraction can be discardedor re-perfused into a patient in a discard or re-perfuse operation 506.

The total number of BMCs, BMC_(total), in the plasma fraction isdetermined in determine operation 508. This operation 508 utilizes atleast one measurement such as a concentration measured by a hemoanalysisapparatus. The BMC concentration, BMC₀, can be multiplied by a volume,V₀, of the liquid, as measured by a flow meter for instance, from whichthe concentration measurement was made. The volume, V₀, times theconcentration, BMC₀, gives a total number of BMCs, BMC_(total). Theplasma fraction is then separated further via second separationoperation 510 (e.g., microfluidic channel separation or centrifugation),in which a BMC-rich plasma fraction (high concentration of BMCs) and aBMC-poor plasma fraction (negligible BMC concentration) are generated.

The BMC-rich fraction and an aliquot, V_(BMC−), of the BMC-poor fractionare mixed in mix operation 512. The volume of the aliquot, V_(BMC−), canbe based on a target BMC concentration, BMCC_(t), and a total number ofBMCs, BMC_(total) (e.g., Equation (7)). The mix operation 512 forms aBMC concentrate which can then be provided to a patient in provide BMCconcentrate to patient operation 516. The remaining BMC-poor fractioncan also be used to form an autologous thrombin preparation forimplantation into a patient in an autologous thrombin preparationoperation 520.

Optionally, after the BMC concentrate is formed in mix operation 512,the BMC concentration can be checked to ensure that the BMCconcentration falls within a target range (or within a margin of errorof a target BMC concentration) in an optional decision 514. If thedecision 514 finds that the BMC concentrate falls within the targetrange or within a margin of error of the target concentration, then theBMC concentrate can be provided to a patient. If not, then theconcentrate can be remixed with the BMC fraction and the process can berepeated starting with the first separation operation 504.

Since settling may cause the concentrate to separate at least partiallyinto a BMC-poor layer and a BMC-rich layer, an agitation operation mayoptionally be used to suspend the BMCs prior to infusion. The method mayalso include an optional filter plasma fraction operation 524 after thefirst separation operation 504. The optional filter operation 526 mayremove any remaining fat and/or large particles. A 170-260 μm filter canbe used in one instance.

FIG. 6 illustrates another method for generating a BMC concentrate of aspecific BMC concentration. The method 600 is nearly identical to themethod 500 with the exception that the determine operation 608 takesplace on the BMC fraction after the second separation operation 610,rather than between the first and second separation operations 604, 610.

FIG. 7 illustrates yet another method for generating a BMC concentrateof a specific BMC concentration. The method 700 is nearly identical tothe methods 500 and 600 with the primary exception that the determineoperation 708 takes place on the whole bone marrow sample, before eitherof the separation operations 704, 710.

Another distinction from the methods 500 and 600 is that the determinetotal number of BMC in bone marrow sample based on at least onemeasurement operation 708 can be carried out in parallel with anoptional add anticoagulant to bone marrow sample operation 722.Alternatively, both operations 708, 722 can be carried out at anyoverlapping or non-overlapping times between the obtain bone marrowsample operation 702 and the first separation operation 704.

A final distinction is that after an optional remix red blood cellfraction with BMC concentrate operation 718, the method 700 may includean optional determine total number of BMCs operation 724. This operation724 can determine a total number of BMCs in the mixture of RBCs and theBMC concentrate after they have been remixed, in the event that the BMCconcentrate does not fall within a target concentration range accordingto decision 714.

FIG. 8 illustrates a block diagram representation of another system 800for generating a target concentration and/or volume of platelets or bonemarrow cells (BMCs). The system 800 obtains or is provided with a wholeblood sample or a BMC sample 802 from a patient 804. The sample canoptionally be mixed with an anticoagulant 806 before entering aseparator 808 (e.g., microfluidic channels or centrifuge, to name two).

The separator 808 can separate the sample 802 into two fractions: a redblood cell (RBC) fraction and a plasma fraction. The separator 808 canalso separate the sample 802 into three fractions: an RBC fraction, atarget cell-poor fraction, and a target cell-rich fraction. Target cellsare the ones that are desired to be in a particular concentration in thefinal concentrate. For instance, platelets, white blood cells, bonemarrow cells, pluripotent cells, and stem cell-like cells are a fewexemplary target cells. Any cell found in a bone marrow sample can be atarget cell. The target cell-poor fraction is one having a negligibleconcentration of target cells while the target cell-rich fraction has agreater-than-natural concentration of target cells.

As each fraction leaves the separator 808, a flow meter 810 can measurea volume of fluid leaving the separator 808. This data can be passed onto a concentration and flow logic and control component 812. Theconcentration and flow logic and control 812 controls a firstcontrollable mixing component 818 in order to control a flow of fluid.The flow logic and control 812 also determines a total number of targetcells by multiplying a concentration of target cells leaving theseparator 808 by a volume of the fluid leaving the separation 808. Theflow logic and control 812 further determines how fluids are to be mixedand in what amounts in order to obtain a target concentration of thetarget cells.

Data representing flow rates, concentrations, volumes, and otherparameters can be displayed to a user via the user interface 814.

The concentration of various particles and cells within each fraction ismeasured by a hemoanalyzer 818. The hemoanalyzer 818 can be embodied ina variety of devices and methods, such as fluorescent activated cellsorting (FACS), optical microscopy, optical light scattering, andelectrical impedance, to name a few. The concentrations measured by thehemoanalyzer 816 are passed to the concentration and flow logic andcontrol 812, which uses these measurements to determine a total numberof target cells in the fluid. From this total number of target cells,the concentration and flow logic and control 812 can determineinstructions for a first controllable mixing component 818 (e.g., pumpor valve or combination of the two).

The first controllable mixing component 818 directs the RBC fractioninto an RBC storage or disposal vessel 820. It also directs thetarget-poor fraction into a target-poor storage or disposal vessel 822.Finally it directs the target-rich fraction into a target-rich storagevessel 824. The order in which these three fractions are directed intotheir respective vessels 820, 822, 824 is not limiting and thus anycombination or order is envisioned.

The concentration and flow logic control 812 also instructs a secondcontrollable mixing component (e.g., pump, valve, or combination of thetwo) to add an aliquot of the target-poor fraction to the entiretarget-rich fraction within the target-rich storage vessel 824. Thevolume of the aliquot is selected so that the combination within thetarget-rich storage vessel 824 has a concentration or concentrationrange of the target cell that meets a target concentration or targetconcentration range.

When this target concentration or concentration range is achieved, atarget cell concentrate exists in the target-rich storage vessel 824 andcan be provided to the patient 804 (or another patient). The remainingtarget-poor fraction in the target-poor storage or disposal vessel 822can also be provide to the patient 804 (or another patient) as anautologous thrombin or a fibrin matrix.

FIG. 9 illustrates a PRP or BMC concentrating apparatus 900. Theapparatus 900 includes a disc centrifuge 902 for separating a wholeblood or BMC sample. The disc centrifuge 902 can separate the sampleinto a red blood cell (RBC) fraction (outside layer), a platelet-richplasma (PRP) or BMC-rich fraction (middle layer), and a platelet-poorplasma (PPP) fraction or BMC-poor fraction (inner layer). The wholeblood or bone marrow sample can be provided to the centrifuge via anopening 918, which can accept the needle of a syringe, for instance. Theopening 918 can be configured such that it is in a closed state unlessthe needle of a syringe is placed into the opening, thus allowing fluidto be passed into the disc centrifuge 902, but not to escape via theopening.

The PPP or BMC-poor fraction can be removed first, and can pass througha flow meter 904 and a hemotology analyzer module 906 to a first vessel910. Fluid can pass through the flow meter 904 or the hemotologyanalyzer module 906 in any order, although as illustrated, flow is firstthrough the flow meter 904. The flow meter 906 provides volume of thePPP or BMC-poor fraction. The removal and flow of the PPP or BMC-poorfraction can be controlled by a computer controlled valve/pump 908.

Once the PPP or BMC-poor fraction has been removed from the disccentrifuge 902, the PRP or BMC-rich fraction becomes the lower orinnermost layer, and can be removed next. The PRP or BMC-rich fractionpasses through the flow meter 906 providing a volume of the PRP orBMC-rich fraction to logic of the apparatus 900 (e.g., a processor). ThePRP or BMC-rich fraction can also pass through a hemotology analyzermodule 906 that measures a total number of platelets or BMCs in the PRPor BMC-rich fraction. Given this volume and total number of platelets orBMCs, logic within the apparatus can determine a concentration ofplatelets or BMCs within the PRP or BMC-rich fraction as the totalnumber of platelets or BMCs times the volume of the PRP or BMC-richfraction. The PRP or BMC-rich fraction can be directed to a secondvessel 912. The removal and flow of the PRP or BMC-rich fraction can becontrolled by the computer controlled valve/pump 908.

The RBC fraction can remain in the disc centrifuge, and since thecentrifuge is disposable, no further action need be taken relative tothe RBC fraction. The logic within the apparatus 900 can determine analiquot of the PPP fraction, or of the BMC-poor fraction, to be mixedwith the PRP or BMC-rich fraction in order to achieve a target plateletor BMC concentration. In one embodiment, a user can set the targetconcentration via a user interface 916 (see FIG. 12). A second computercontrolled valve/pump 914 can allow the aliquot to pass from the firstvessel 910 to the second vessel 912 in order to form the PRP or BMCconcentrate. An optional agitation mechanism (not illustrated) may beactivated to improve mixing of the PRP or BMC concentrate within thesecond vessel 912.

The remaining PPP fraction or BMC-poor fraction and the PRP or BMCconcentrate can be removed from the vessels 910, 912 via respectiveopenings 920 and 922, which may be accessible via the needle of asyringe in one embodiment (see FIG. 13). At the same time, the vessels910, 912 may be separable and removable such that they can each be movedto a patient or storage location for later use.

Although FIG. 9 has been described where fluid is removed from the disccentrifuge 902 from a middle of the centrifuge 902, in otherembodiments, fluid can leave the disc centrifuge from other points. Forinstance, fluid can leave from an outer radius of the centrifuge in someembodiments. Also, the order of removing different fractions can bevaried. In some embodiments, a printer 924 can provide hardcopies ofdata from the apparatus 900.

To ensure sterility, those portions of the apparatus 900 that contactblood or bone marrow can be modular and disposable. FIG. 10 illustratesone embodiment of a package 1000 comprising those components of theapparatus 900 that can be disposable. These can include any one or moreof the following illustrated components: the centrifuge 902, thecentrifuge opening 918, the hematology analyzer module 906, the firstcomputer controlled valve/pump 908, and the first and second vessels910, 912. Additionally, the disposable portions of the apparatus 900 caninclude a fluid path 924 between the centrifuge 902 and thecomputer-controlled valve/pump 908, and a fluid path 926 connecting thefirst and second vessels 910, 912. Any two or more of these componentscan be interlinked to simplify and ease installation, removal, andtransport, and the interlinked package 1000 can be replaced by a similaror identical package 1000.

FIG. 11 illustrates how a syringe 928 can be used to provide a wholeblood or bone marrow sample to the apparatus 900 of FIG. 9 via insertionthrough the opening 918.

FIG. 12 illustrates a user interacting with the apparatus 900 of FIG. 9,for instance, via a touchscreen embodiment of the user interface 916.

FIG. 13 illustrates how a syringe 930 can be used to remove contents ofthe first vessel 910 illustrated in FIGS. 9-13. As described, the firstvessel 910 can hold a PPP fraction or a BMC-poor fraction, and thesyringe can be used to extract a part or all of the contents of thefirst vessel 910.

While FIGS. 9-13 have described the first vessel 910 as typicallystoring the PPP or BMC-poor fraction, and the second vessel 912 asstoring the PRP or BMC-rich fraction or PRP or BMC concentrate, one ofskill in the art will recognize that the two vessels 910, 912 areinterchangeable and thus not limited to a right or left of the apparatus900.

The systems and methods described herein can be implemented in a machinesuch as a computer system in addition to the specific physical devicesdescribed herein. FIG. 14 shows a diagrammatic representation of oneembodiment of a machine in the exemplary form of a computer system 1400within which a set of instructions can execute for causing a device toperform or execute any one or more of the aspects and/or methodologiesof the present disclosure. The components in FIG. 14 are examples onlyand do not limit the scope of use or functionality of any hardware,software, embedded logic component, or a combination of two or more suchcomponents implementing particular embodiments.

Computer system 1400 may include a processor 1401, a memory 1403, and astorage 1408 that communicate with each other, and with othercomponents, via a bus 1440. The bus 1440 may also link a display 1432,one or more input devices 1433 (which may, for example, include akeypad, a keyboard, a mouse, a stylus, etc.), one or more output devices1434, one or more storage devices 1435, and various tangible storagemedia 1436. All of these elements may interface directly or via one ormore interfaces or adaptors to the bus 1440. For instance, the varioustangible storage media 1436 can interface with the bus 1440 via storagemedium interface 1426. Computer system 1400 may have any suitablephysical form, including but not limited to one or more integratedcircuits (ICs), printed circuit boards (PCBs), mobile handheld devices(such as mobile telephones or PDAs), laptop or notebook computers,distributed computer systems, computing grids, or servers.

Processor(s) 1401 (or central processing unit(s) (CPU(s))) optionallycontains a cache memory unit 1402 for temporary local storage ofinstructions, data, or computer addresses. Processor(s) 1401 areconfigured to assist in execution of computer readable instructions.Computer system 1400 may provide functionality as a result of theprocessor(s) 1401 executing software embodied in one or more tangiblecomputer-readable storage media, such as memory 1403, storage 1408,storage devices 1435, and/or storage medium 1436. The computer-readablemedia may store software that implements particular embodiments, andprocessor(s) 1401 may execute the software. Memory 1403 may read thesoftware from one or more other computer-readable media (such as massstorage device(s) 1435, 1436) or from one or more other sources througha suitable interface, such as network interface 1420. The software maycause processor(s) 1401 to carry out one or more processes or one ormore steps of one or more processes described or illustrated herein.Carrying out such processes or steps may include defining datastructures stored in memory 1403 and modifying the data structures asdirected by the software.

The memory 1403 may include various components (e.g., machine readablemedia) including, but not limited to, a random access memory component(e.g., RAM 1404) (e.g., a static RAM “SRAM”, a dynamic RAM “DRAM”,etc.), a read-only component (e.g., ROM 1405), and any combinationsthereof. ROM 1405 may act to communicate data and instructionsunidirectionally to processor(s) 1401, and RAM 1404 may act tocommunicate data and instructions bidirectionally with processor(s)1401. ROM 1405 and RAM 1404 may include any suitable tangiblecomputer-readable media described below. In one example, a basicinput/output system 1406 (BIOS), including basic routines that help totransfer information between elements within computer system 1400, suchas during start-up, may be stored in the memory 1403.

Fixed storage 1408 is connected bidirectionally to processor(s) 1401,optionally through storage control unit 1407. Fixed storage 1408provides additional data storage capacity and may also include anysuitable tangible computer-readable media described herein. Storage 1408may be used to store operating system 1409, EXECs 1410 (executables),data 1411, API applications 1412 (application programs), and the like.Often, although not always, storage 1408 is a secondary storage medium(such as a hard disk) that is slower than primary storage (e.g., memory1403). Storage 1408 can also include an optical disk drive, asolid-state memory device (e.g., flash-based systems), or a combinationof any of the above. Information in storage 1408 may, in appropriatecases, be incorporated as virtual memory in memory 1403.

In one example, storage device(s) 1435 may be removably interfaced withcomputer system 1400 (e.g., via an external port connector (not shown))via a storage device interface 1425. Particularly, storage device(s)1435 and an associated machine-readable medium may provide nonvolatileand/or volatile storage of machine-readable instructions, datastructures, program modules, and/or other data for the computer system1400. In one example, software may reside, completely or partially,within a machine-readable medium on storage device(s) 1435. In anotherexample, software may reside, completely or partially, withinprocessor(s) 1401.

Bus 1440 connects a wide variety of subsystems. Herein, reference to abus may encompass one or more digital signal lines serving a commonfunction, where appropriate. Bus 1440 may be any of several types of busstructures including, but not limited to, a memory bus, a memorycontroller, a peripheral bus, a local bus, and any combinations thereof,using any of a variety of bus architectures. As an example and not byway of limitation, such architectures include an Industry StandardArchitecture (ISA) bus, an Enhanced ISA (EISA) bus, a Micro ChannelArchitecture (MCA) bus, a Video Electronics Standards Association localbus (VLB), a Peripheral Component Interconnect (PCI) bus, a PCI-Express(PCI-X) bus, an Accelerated Graphics Port (AGP) bus, HyperTransport(HTX) bus, serial advanced technology attachment (SATA) bus, and anycombinations thereof.

Computer system 1400 may also include an input device 1433. In oneexample, a user of computer system 1400 may enter commands and/or otherinformation into computer system 1400 via input device(s) 1433. Examplesof an input device(s) 1433 include, but are not limited to, analpha-numeric input device (e.g., a keyboard), a pointing device (e.g.,a mouse or touchpad), a touchpad, a joystick, a gamepad, an audio inputdevice (e.g., a microphone, a voice response system, etc.), an opticalscanner, a video or still image capture device (e.g., a camera), and anycombinations thereof. Input device(s) 1433 may be interfaced to bus 1440via any of a variety of input interfaces 1423 (e.g., input interface1423) including, but not limited to, serial, parallel, game port, USB,FIREWIRE, a THUNDERBOLT hardware interface (THUNDERBOLT is a registeredtrademark of Intel Corporation), or any combination of the above.

In particular embodiments, when computer system 1400 is connected tonetwork 1430, computer system 1400 may communicate with other devices,specifically mobile devices and enterprise systems, connected to network1430. Communications to and from computer system 1400 may be sentthrough network interface 1420. For example, network interface 1420 mayreceive incoming communications (such as requests or responses fromother devices) in the form of one or more packets (such as InternetProtocol (IP) packets) from network 1430, and computer system 1400 maystore the incoming communications in memory 1403 for processing.Computer system 1400 may similarly store outgoing communications (suchas requests or responses to other devices) in the form of one or morepackets in memory 1403 and communicated to network 1430 from networkinterface 1420. Processor(s) 1401 may access these communication packetsstored in memory 1403 for processing.

Examples of the network interface 1420 include, but are not limited to,a network interface card, a modem, and any combination thereof. Examplesof a network 1430 or network segment 1430 include, but are not limitedto, a wide area network (WAN) (e.g., the Internet, an enterprisenetwork), a local area network (LAN) (e.g., a network associated with anoffice, a building, a campus or other relatively small geographicspace), a telephone network, a direct connection between two computingdevices, and any combinations thereof. A network, such as network 1430,may employ a wired and/or a wireless mode of communication. In general,any network topology may be used.

Information and data can be displayed through a display 1432. Examplesof a display 1432 include, but are not limited to, a liquid crystaldisplay (LCD), an organic liquid crystal display (OLED), a cathode raytube (CRT), a plasma display, and any combinations thereof. The display1432 can interface to the processor(s) 1401, memory 1403, and fixedstorage 1408, as well as other devices, such as input device(s) 1433,via the bus 1440. The display 1432 is linked to the bus 1440 via a videointerface 1422, and transport of data between the display 1432 and thebus 1440 can be controlled via the graphics control 1421.

In addition to a display 1432, computer system 1400 may include one ormore other peripheral output devices 1434 including, but not limited to,an audio speaker, a printer, and any combinations thereof. Suchperipheral output devices may be connected to the bus 1440 via an outputinterface 1424. Examples of an output interface 1424 include, but arenot limited to, a serial port, a parallel connection, a USB port, aFIREWIRE port, a THUNDERBOLT port, and any combinations thereof.

In addition or as an alternative, computer system 1400 may providefunctionality as a result of logic hardwired or otherwise embodied in acircuit, which may operate in place of or together with software toexecute one or more processes or one or more steps of one or moreprocesses described or illustrated herein. Reference to software in thisdisclosure may encompass logic, and reference to logic may encompasssoftware. Moreover, reference to a computer-readable medium mayencompass a circuit (such as an IC) storing software for execution, acircuit embodying logic for execution, or both, where appropriate. Thepresent disclosure encompasses any suitable combination of hardware,software, or both.

Those of skill in the art would understand that information and signalsmay be represented using any of a variety of different technologies andtechniques. For example, data, instructions, commands, information,signals, bits, symbols, and chips that may be referenced throughout theabove description may be represented by voltages, currents,electromagnetic waves, magnetic fields or particles, optical fields orparticles, or any combination thereof.

Those of skill would further appreciate that the various illustrativelogical blocks, modules, circuits, and algorithm steps described inconnection with the embodiments disclosed herein may be implemented aselectronic hardware, computer software, or combinations of both. Toclearly illustrate this interchangeability of hardware and software,various illustrative components, blocks, modules, circuits, and stepshave been described above generally in terms of their functionality.Whether such functionality is implemented as hardware or softwaredepends upon the particular application and design constraints imposedon the overall system. Skilled artisans may implement the describedfunctionality in varying ways for each particular application, but suchimplementation decisions should not be interpreted as causing adeparture from the scope of the present invention.

The various illustrative logical blocks, modules, and circuits describedin connection with the embodiments disclosed herein may be implementedor performed with a general purpose processor, a digital signalprocessor (DSP), an application specific integrated circuit (ASIC), afield programmable gate array (FPGA) or other programmable logic device,discrete gate or transistor logic, discrete hardware components, or anycombination thereof designed to perform the functions described herein.A general purpose processor may be a microprocessor, but in thealternative, the processor may be any conventional processor,controller, microcontroller, or state machine. A processor may also beimplemented as a combination of computing devices, e.g., a combinationof a DSP and a microprocessor, a plurality of microprocessors, one ormore microprocessors in conjunction with a DSP core, or any other suchconfiguration.

The steps of a method or algorithm described in connection with theembodiments disclosed herein may be embodied directly in hardware, in asoftware module executed by a processor, or in a combination of the two.A software module may reside in RAM memory, flash memory, ROM memory,EPROM memory, EEPROM memory, registers, hard disk, a removable disk, aCD-ROM, or any other form of storage medium known in the art. Anexemplary storage medium is coupled to the processor such the processorcan read information from, and write information to, the storage medium.In the alternative, the storage medium may be integral to the processor.The processor and the storage medium may reside in an ASIC. The ASIC mayreside in a user terminal. In the alternative, the processor and thestorage medium may reside as discrete components in a user terminal.

In conclusion, the present invention provides, among other things,systems and methods that autonomously or semi-autonomously produce a PRPor BMC concentrate having a target concentration of platelets or BMCs.Those skilled in the art can readily recognize that numerous variationsand substitutions may be made in the invention, its use, and itsconfiguration to achieve substantially the same results as achieved bythe embodiments described herein. For instance, blood products can bemoved through the system 100 and 800, or to other systems, eithermanually or autonomously. As another example, methods for separatingblood components besides centrifugation can be used (e.g., microfluidicchannel separation or electrical impedance separation). Accordingly,there is no intention to limit the invention to the disclosed exemplaryforms. Many variations, modifications, and alternative constructionsfall within the scope and spirit of the disclosed invention.

What is claimed is:
 1. A cell concentrating system comprising: a blood separation component having a blood sample input and having a centrifuge to separate the blood sample into a red blood cell fraction and a plasma fraction, the plasma fraction comprising a target cell-rich fraction and a target cell-poor fraction, the blood sample having a volume of between 60 mL and no more than 250 mL; a first vessel for storing the target cell-poor fraction separated from the blood sample; a second vessel for storing the target cell-rich fraction separated from the blood sample; a first valve upstream of the first vessel and the second vessel, and downstream of the blood separation component; a second valve for providing selective fluid communication between the first vessel and the second vessel; a concentration and flow logic and control component configured to: control the first valve to remove the target cell-poor fraction to the first vessel and to remove the target cell-rich fraction to the second vessel; determine a first volume of the target cell-poor fraction in the first vessel to mix with the target cell-rich fraction in the second vessel in order to form a target cell-rich concentrate having a concentration of target cells that is within a target concentration range for studying or defining a dose-response relationship in a patient; determine whether the target cell-rich concentrate has a concentration of target cells within the target concentration range; and control the second valve to transfer the first volume of the target cell-rich fraction from the first vessel to the second vessel to form the target cell-rich concentrate; wherein the target concentration range is between 1.0 and 1.5×10⁶ target cells/μL; and the cell concentrating system is autologous.
 2. The cell concentrating system of claim 1, wherein the blood sample is a whole blood sample or a bone marrow sample.
 3. The cell concentrating system of claim 1, further comprising a measurement system, wherein the measurement system comprises: a concentration measurement component that measures a concentration of target cells in the autologous cell concentrating system; and a flow meter component that measures a second volume in which the concentration was measured.
 4. The cell concentrating system of claim 3, wherein the concentration measurement component measures a concentration of target cells in the target cell-rich fraction and the second volume is that of the target cell-rich fraction.
 5. The cell concentrating system of claim 3, wherein the concentration measurement component is configured to perform a function selected from the group consisting of: optical microscopy, optical light scattering, and electrical impedance.
 6. The cell concentrating system of claim 1, wherein the blood separation component is configured to perform a function selected from the group consisting of: centrifugation, laser impedance, flow cytometry, dielectrophoresis, micro fluidic channel separation, electrical impedance, and use of fluorescent markers.
 7. The cell concentrating system of claim 1, wherein the target cells are selected from the group consisting of: pluripotent cells, white blood cells, red blood cells, platelets, Neutrophils, Monocytes, Lymphoctyes, Eosinophils, and Basophils.
 8. A method of generating a target cell-rich concentrate comprising: obtaining a target blood sample having a volume of between 60 mL and 250 mL; inputting the target blood sample into a cell concentration system; causing the cell concentration system to: separate the target blood sample into a red blood cell fraction, a target cell-poor fraction, and a target cell-rich fraction; remove the target cell-poor fraction to a first vessel; remove the target cell-rich fraction to a second vessel in selective fluid communication with the first vessel; determine a first volume of the target cell-poor fraction in the first vessel to mix with the target cell-rich fraction in the second vessel in order to form a target cell-rich concentrate having a concentration of target cells that is within a target concentration range for studying or defining a dose-response relationship in a patient; determine whether the target cell-rich concentrate has a concentration of target cells within the target concentration range; and transfer the first volume of the target cell-rich fraction from the first vessel to the second vessel to form the target cell-rich concentrate; wherein the target concentration range is between 1.0 and 1.5×106 target cells/μL; and the target cell-rich concentrate is an autologous target cell-rich concentrate of the target blood sample.
 9. The method of claim 8, wherein the target blood cell is selected from the group consisting of: pluripotent cells, white blood cells, red blood cells, platelets, Neutrophils, Monocytes, Lymphoctyes, Eosinophils, and Basophils.
 10. The method of claim 8, wherein the target blood sample is a whole blood sample or a bone marrow sample.
 11. The method of claim 8, further comprising: causing the cell concentration system to: determine the target cell concentration before or after the separating.
 12. The method of claim 11, wherein the separating includes a first and second separating, and wherein the determining the target cell concentration is performed between the first and second separating.
 13. A method of autologous therapeutics, comprising: extracting a target blood sample from a patient, the target blood sample having a volume of between 60 mL and 250 mL; inputting the target blood sample into a cell concentration system; causing the cell concentration system to: separate the target blood sample into a red blood cell fraction, a target cell-poor fraction, and a target cell-rich fraction; remove the target cell-poor fraction to a first vessel; remove the target cell-rich fraction to a second vessel in selective fluid communication with the first vessel; determine a first volume of the target cell-poor fraction in the first vessel to mix with the target cell-rich fraction in the second vessel in order to form a target cell-rich concentrate having a concentration of target cells that is within a target concentration range for studying or defining a dose-response relationship in a patient; determine whether the target cell-rich concentrate has a concentration of target cells within the target concentration range; and transfer the first volume of the target cell-rich fraction from the first vessel to the second vessel to form the target cell-rich concentrate; wherein the target concentration range is between 1.0 and 1.5×106 target cells/μL; and the target cell-rich concentrate is an autologous target cell-rich concentrate of the target blood sample; and injecting the autologous target cell-rich concentrate into the patient.
 14. The method of claim 13, wherein the target blood cell is selected from the group consisting of: pluripotent cells, white blood cells, red blood cells, platelets, Neutrophils, Monocytes, Lymphoctyes, Eosinophils, and Basophils.
 15. The method of claim 13, wherein the target blood sample is a whole blood sample or a bone marrow sample.
 16. The method of claim 13, further comprising: causing the cell concentration system to determine the target cell concentration before or after the separating.
 17. The method of claim 16, wherein the separating includes a first and second separating, and wherein the determining the target cell concentration is performed between the first and second separating. 